The described invention relates to compounds and methods for covalently attaching a label to a target molecule. More specifically, the compounds are alkylating compounds having a reporter molecule and the covalent attachment is performed by a one-step alkylation reaction.
The analysis and detection of minute quantities of substances in biological and non-biological samples has become a routine practice in clinical and analytical laboratories. These detection techniques can be divided into two major classes: (1) those based on ligand-receptor interactions (e.g., immunoassay-based techniques) and (2) those based on nucleic acid hybridization (polynucleotide sequence-based techniques).
Immunoassay-based techniques are characterized by a sequence of steps comprising the non-covalent binding of an antibody and an antigen complementary to it. Polynucleotide sequence-based detection techniques have been characterized by a sequence of steps comprising the non-covalent binding of a labeled polynucleotide sequence or probe to a complementary sequence of the analyte under conditions which permit hybridization of the bases through Watson-Crick pairing, and the detection of that hybridization.
The non-covalent binding of a labeled sequence or probe to a complementary sequence of a nucleic acid is the primary recognition event of polynucleotide sequence-based detection techniques. This binding event is brought about by a precise molecular alignment and interaction of complementary nucleotides of the probe and target. It is energetically favored by the release of non-covalent bonding free energy, e.g., hydrogen bonding, stacking free energy and the like.
In order to employ the non-covalent binding of a probe for the determination of a nucleic acid containing a target sequence, it is necessary to be able to detect binding of the probe to the target. This detection is effected through a signaling step or event. A signaling step or event allows detection in some quantitative or qualitative manner of the occurrence of the primary recognition event.
A wide variety of signaling events may be employed to detect the occurrence of the primary recognition event. The signaling event chosen depends on the particular signal that characterizes the reporter molecule employed. Although the labeling reagent itself, without further treatment, may be detectable, more often, either the reporter molecule is attached covalently, or bound non-covalently to a labeling reagent.
There are a wide variety of reporter molecules that may be employed for covalent attachment to the labeling reagent of polynucleotide sequences useful as probes in nucleic acid detection systems. All that is required is that the reporter molecule provide a signal that may be detected by appropriate means and that has the ability to attach covalently to the labeling reagent.
Reporter molecules may be radioactive or non-radioactive. Radioactive signaling moieties are characterized by one or more radioisotopes of phosphorous, iodine, hydrogen, carbon, cobalt, nickel, and the like. Preferably the radioisotope emits .beta. or .gamma. radiation, and has a long half-life. Detection of radioactive reporter molecules is typically accomplished by the stimulation of photon emission from crystalline detectors caused by the radiation, or by the fogging of a photographic emulsion.
Non-radioactive reporter molecules have the advantage that their use does not pose the hazards associated with exposure to radiation, and that special disposal techniques after use are not required. In addition, they are generally more stable, and as a consequence, cheaper to use. Detection sensitivities of non-radioactive reporter molecules may be as high or higher than those of radioactive reporter molecules.
The ability to label DNA with a non-radioactive detectable marker simply and reliably makes it attractive for use in a wide variety of molecular and cellular biology applications. Some specific applications in which a non-radioactively labeled DNA or RNA probe can be used include hybridization reaction procedures (southern, northern, slot, or dot blots, in situ hybridization), nucleic acid localization studies, DNA or RNA quantitation and DNase or RNase quantitation.
Both enzyme mediated and direct labeling protocols have been developed to attach non-radioactive detectable tags such as the fluorescent compounds fluorescein and rhodamine (and others) to DNA. While these labeling methods have allowed non-radioactive detection systems to approach or even surpass the radioactive methods in terms of sensitivity there remains significant disadvantages with each of the non-radioactive labeling systems developed to date. 1) Enzymatic DNA labeling systems require a number of reagents including both unlabeled and labeled nucleotide precursors, primers, and/or enzymes to facilitate DNA synthesis. Labeling efficiency is not easily controlled and for the two most common labeling reactions (nick translation and random priming) it is not possible to create a labeled probe that is the same size as the starting DNA. 2) Direct labeling methods also have significant limitations which include a lower efficiency of labeling resulting in reduced sensitivity, laborious multi-step labeling protocols, harsh reaction conditions, variability from reaction to reaction, and unstable reactants.
Direct labeling methods have been developed for chemically modifying nucleic acids for use as detectable probes in hybridization experiments.
Sodium bisulfite may be used in the presence of a diamine to introduce primary amines on cytosine residues which could then be subsequently modifed with a reporter group. Adarichev et al (Adarichev, V. A.; Vorobeva, N. V.; Grafodatskii, A. S.; Dymshits, G. M.; and Sablina, O. V. (1995) Molecular Biology 29(3): 538-545) used 4-aminohydroxybutylamine to transaminate cytosine residues in a similar fashion. DNA has been modified at the C-8 position of adenine or guanine using a diazonium salt attached to biotin. The diazonium salt is generated in-situ with sodium nitrite then directly reacted with DNA. In another labeling procedure, the carcinogen 2-acetylaminofluorene was modified to the reactive compound N-acetoxy-2-acetylaminofluorene by Landegent et al and attached to the C-8 position of guanine. DNA modified by this reagent was subsequently detected using antibodies directed against the modified guanosine. The reactive aldehyde at the C8 position (N7-formyl group) of a ring-opened guanine has also been used as a target for direct labeling using an aldehyde reactive nucleophile such as hydrazine attached to a detectable label.
These reagents have different limitations, some of these limitations are multi-step synthesis, the ability to derivatize only single stranded DNA, the need to use large amounts of reagent or other harsh conditions to get adequate amounts of DNA modification, and the modification of amines involved in double-stranded DNA base pairing.
The techniques of Northern and Southern blotting are two of the most powerful and frequently used procedures in molecular biology. Yet the necessary manipulations are time consuming and are not likely to be automated under current technology. Often the polynucleotide (RNA, DNA) under analysis must first be fractionated by size, transferred onto a solid support and then treated through a series of steps to ensure only specific binding of a probe. Detection of the hybridized products usually depends on radiolabeling, heavy metal derivatization or antibody complexation. The methods of blotting have been a staple of basic research, and now also serve in an ever increasing number of commercial kits used to diagnose genetic, malignant, and infectious diseases.
In 1967 Belikova et al. (Belikova, A. M., Zarytova, V. F. and Grineva, N. I. (1967) Tetrahedron Letters, 37:3557-62) first described monoadduct alkylation of ribonucleosides and diribonucleoside phosphates using 2-chloroethylamine residues. While this work provided evidence that ribonucleosides could be covalently modified with the alkylating mustard derivative, the efficiency of the process was very low. Utilizing a multi-step process Frumgarts et al. (Frumgarts, L. A.; Kipriyanov, S. M.; Kalachikov, S. M.; Dudareva, N. A.; Dymshits, G. M.; Karpova, G. G.; and Salganik, R. I. (1986) Bioorg. Khim. 12(11): 1508-1513) alkylated DNA using the nitrogen mustard 4-(N-methylamino-N-2-chloroethyl)benzylamine, and subsequently attached fluorescent labels to the amine that had been covalently attached to the DNA. This multi-step process required that the mustard and fluorescent label be used in a large molar excess to the DNA being labeled.
Quinicrine (acridine) is a DNA intercalating molecule which is also fluorescent. Caspersson et al. used this molecule both with and without the attachment of a nitrogen mustard to obtain chemical and physicochemical information about metaphase chromosomal structure. In this study, the fluorescent pattern obtained using quinicrine, which contains no alkylating group, produced a band pattern of the same type as the quinicrine mustard. (Caspersson,T.; Zech, L.; Modest, E. J.; Foley, G. E.; Wagh, U.; and Simonsson, (1969) E. Experimental Cell Research 58 128-140).
Utilizing the nucleic acid alkylating ability of the nitrogen mustards we have developed a series of labeling reagents consisting of 1) a nitrogen mustard moiety, 2) a cationic linker and 3) a detectable marker. A nucleic acid reactive nitrogen mustard derivative used in the synthesis of these labeling agents can be the aromatic nitrogen mustard 4-[(2-chloroethyl)-methylamino]-benzaldehyde. This nitrogen mustard derivative was described in U.S. Pat. No. 2,141,090.
The ideal labeling method would combine one step simplicity with high efficiency labeling that results in a labeled product that remains intact, stable and the same size as the starting DNA. To develop a labeling reagent with these characteristics we used nitrogen mustards having the ability to alkylate DNA in a one step reaction resulting in DNA containing a covalently attached adduct.
The procedure for labeling results in the formation of a chemical bond between the compound and the polynucleic acid and/or protein. The labeling procedure consists of incubating the polynucleic acid with the said compounds in aqueous or non-aqueous solutions, followed by separation of the labeled polynucleic acid from the unreacted labeling reagent. The extent of labeling can be controlled by regulating the relative amounts of labeling reagent and polynucleic acid, by adjusting the length of the incubation, by controlling the temperature of the incubation, by controlling the absolute concentrations of polynucleic acid and labeling reagent, and by controlling the composition of the aqueous or organic solution using solvent, pH, ionic strength, and buffers.
The labeled polynucleic acid can be used for several purposes:
1) techniques to detect specific sequences of polynucleic acids that rely upon hybridization or binding affinity of the labeled polynucleic acid to target nucleic acid or protein; including dot blots, slot blots, Southern blots, Northern blots, Southwestern blot, FISH (fluorescent in situ hybridization), in situ hybridization of RNA and DNA sequences, and newly developing combinatorial techniques in which the polynucleic acid is on a xe2x80x9cchipxe2x80x9d or multiwell or multislot device.
2) labeling polynucleic acids that are delivered to cells in vitro or in vivo so as to determine their sub-cellular and tissue location
3) labeling oligonucleotides that are used as primer in amplification techniques such as PCR (polymerase chain reaction)
4) quantitating polynucleic acids
5) quantitating nucleases (including RNases and DNases) by fluorescence polarization or fluorescence dequenching.
6) sequencing polynucleic acids
7) directly detecting mutations
8) covalently attaching reactive groups for use in anti-sense applications.
Also described is a method of functionalizing digoxin. Digoxin is functionalized by first oxidizing the diol functionality on the terminal sugar residue forming digoxin di-aldehyde, and subsequent reductive alkylation using sodium cyanoborohydride and a linking compound. The linker consists of an organic molecule with a primary amine and a protected primary amine, which after removal of the protecting group results in a digoxin derivative with a primary amino group. The primary amine can be transformed into a thiol reactive group such as a haloacetamide, a maleimide, a disulfide, or a pyridyldithio; an aldehyde or ketone reactive group such as a thiosemicarbazide or a hydrazide; an amine reactive group such as a succinimidyl ester, isothiocyanate, or sulfonyl chloride; or a carboxylic acid reactive group such as a diazomethane or a haloacetyl. This methodology results in a digoxin molecule that can be used to label proteins, peptides, polysaccharides, lipids, and other molecules of interest. The straight-forward synthesis from inexpensive precursors also lends utility to this unique method of supplying a digoxin label.
A method is provided for a single-step covalent attachment of a label to a nucleic acid comprising forming a covalently attachable labeling reagent for alkylating the nucleic acid. Then, combining the covalently attachable labeling reagent with a mixture containing the nucleic acid, under conditions wherein the labeling reagent has reactivity with the nucleic acid thereby forming a covalent bond.
A compound is provided that is a labeling molecule comprising a covalently attachable labeling reagent for alkylating a chemical in a single-step reaction, thereby providing the chemical with a detectable reporter molecule.
In a preferred embodiment, a compound is provided having the structure comprising
Dxe2x80x94Bxe2x80x94A
wherein, D is selected from the group consisting of fluorescence-emitting compounds, radioactive compounds, haptens, immunogenic molecules, chemiluminescence-emitting compounds, and proteins; B is selected from the group of molecules having an affinity for nucleic acids by interactions consisting of electrostatic, minor groove binding, major groove binding, and intercalation; and, A is selected from the group of alkylating agents consisting of mustards and three-membered ring derivatives.
In a preferred embodiment, a compound having the general structure comprising 
wherein, D is selected from the group consisting of fluorescence-emitting compounds, radioactive compounds, haptens, immunogenic molecules, chemiluminescence-emitting compounds, and proteins; R is selected from the group of alkyls and hydrogen; Rxe2x80x2 is selected from the group of alkyls and hydrogen; n is an integer from 1 to 20; m is an integer from 1 to 20; x is an integer from 1 to 5; and, A is selected from the group of alkylating agents consisting of mustards and three-membered ring derivatives.
Also provided is a kit comprising a receptacle containing a covalently attachable labeling reagent for alkylating a chemical in a single-step reaction. Instructions for use are also provided with the kit. By the term instructions for use, it is meant a tangible expression describing the reagent concentration for at least one assay method, parameters such as the relative amount of reagent and sample to be admixed, maintenance time periods for reagent/sample admixtures, temperature, buffer conditions and the like.
Reference is now made in detail to the preferred embodiments of the invention, examples of which are illustrated in the accompanying drawings.